Advanced Techniques in Diagnostic Microbiology

20. Analysis of 16S rRNA Gene Sequence 325
Methodology
Four steps are required to reach bacterial identification through 16S rDNA sequencing:
DNA extraction, PCR amplification, nucleotide sequencing, and database
homology search and reporting.
DNA Extraction
Pure culture of a bacterium is generally required for its identification. One or
two isolated colonies or a visible pellet from pure liquid culture contain up to
10 10 cells, and the extracted DNA will be sufficient for many subsequent PCRs.
Various DNA extraction methods can be used, such as traditional phenol chloroform
method, commercial DNA extraction kits, and so forth. Pure culture and
relatively large quantity of target DNA makes contamination by background DNA
from reagents and other sources almost negligible. Because the extracted DNA
will be amplified by PCR, its purity requirement is not strict either. Therefore,
for routine clinical application, the simpler and quicker the method is, the better.
Here is a simple method used in our laboratory (Han et al., 2002). Two
colonies or the pellet of 1 mL positive liquid medium (upon confirmation of purity
by staining) are resuspended in 200 μL extraction solution (Prepman Ultra;)
(Applied Biosystems, Foster City, CA, USA). The suspension is boiled for 10
min and centrifuged for 5 min at 8000 × g, and the genomic DNA is extracted
in the supernatant. One percent of the supernatant (2 μL) is used for subsequent
PCR.
PCR
The amplification by PCR is no different from other PCR methods. Thermal cycles
and conditions vary slightly depending on polymerase and primers. PCR product
can be examined and the amount estimated visually on an agarose gel electrophoresis.
This step, however, being intermediate, is not essential. In our experience with
universal primers and mycobacteria-specific primers (Han et al., 2002), PCR are
robust and yield sufficient amplicons for sequencing to obviate the electrophoresis
step.
Nucleotide Sequencing
The PCR amplicon can be sequenced directly after removal of unpolymerized
primers and deoxynucleoside triphosphates that is achieved by enzymatic digestion
with exonuclease and shrimp alkaline phosphatase. No further purification or
concentration of the amplicon is generally necessary. Automated sequencing can
be completed in no more than a few hours.